title: Modulation of the UGT2B7 enzyme activity by C-terminally truncated proteins derived from alternative splicing.
abstract: The enzyme UGT2B7 is one of the most active UDP-glucuronosyltransferases (UGTs) involved in drug metabolism and in maintaining homeostasis of endogenous compounds. We recently reported the existence of 22 UGT2B7 mRNAs, two with a classic 5' region but alternative 3' ends namely UGT2B7_v5 (containing a novel terminal exon 6b) and _v7 (exon 5 excluded) that encode enzymatically inactive isoforms 2 and 4 (i2 and i4), respectively. The v1 mRNA encoding the UGT2B7 enzyme (renamed isoform 1 or i1) is coexpressed with the splice variants v5 and v7 in human liver, kidney, and small intestine and the hepatic cell lines HepG2 and C3A. The presence of alternate v5 and v7 transcripts in isolated polysomes from these hepatic cells further supports endogenous protein translation. Cellular fractionation of clonal HEK293 cell lines overexpressing UGT2B7 isoforms demonstrates that i1, i2, and i4 proteins colocalize in the microsomal/Golgi fraction, whereas i2 and i4 can also be found in the cytosol; a finding sustained by immunofluorescence experiments using tagged proteins. By modifying splice variant abundance in overexpression in HEK293 and HepG2 cells as well as RNA interference experiments in HepG2 and C3A cells, we observe drug glucuronidation phenotypes compatible with variant-mediated repression of UGT2B7 activity without consequent alteration of the apparent enzyme affinity (K(m)). Finally, coimmunoprecipitation experiments support a direct protein-protein interaction of i2 and i4 proteins with the functional UGT2B7 enzyme as a potential causative mechanism. These findings point toward a novel autoregulatory mechanism of the UGT2B7 glucuronidation pathway by naturally occurring alternative i2 and i4 proteins.
authors.affiliation: Pharmacogenomics Laboratory, Centre Hospitalier de l'Université Laval de Québec (CHU de Québec) Research Center, Faculty of Pharmacy, Laval University, Québec, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Ménard
authors.forename: Pierre
authors.initials: P
authors.lastname: Collin
authors.forename: Guillaume
authors.initials: G
authors.lastname: Margaillan
authors.forename: Chantal
authors.initials: C
authors.lastname: Guillemette
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007421:Intestine, Small / Q000201:enzymology / Q000378:metabolism; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D012333:RNA, Messenger; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase